Cross-reactivity of cell-selective CRRETAWAC peptide with human and porcine endothelial cells.

We report on the cross-reactivity of the cell adhesive peptide CRRETAWAC between human and porcine endothelial cells (ECs). CRRETAWAC is a phage display derived peptide which has been shown to bind the α5 ß1 receptor on human ECs, but does not bind platelets and thus could be incorporated into a coating for cardiovascular biomaterials that resists platelet adhesion and thrombosis, while allowing for endothelialization. To determine the cross-reactivity of the peptide, attachment and growth of human and porcine ECs on CRRETAWAC fluorosurfactant polymer (FSP) coated surfaces was explored. CRRETAWAC FSP was synthesized and characterized by mass spectrometry, NMR, and IR spectroscopy. pEC attachment and growth on CRRETAWAC FSP was similar to the positive controls, human fibronectin and RGD FSP, achieving confluence in 72 h. Initial adhesion on CRRETAWAC FSP was also similar for porcine and human ECs. Blocking with soluble CRRETAWAC peptide reduced adhesion to CRRETAWAC coated surfaces by over 50%, indicating that the pECs specifically bind CRRETAWAC peptide. With this study, we have demonstrated that CRRETAWAC peptide coated surfaces are capable of binding porcine ECs in a specific manner and supporting a confluent layer of pECs. In vitro validation of the porcine model was critical for ensuring the best chance of success for the in vivo testing of CRRETAWAC coated ePTFE vascular grafts.

Endothelial cell attachment and shear response on biomimetic polymer-coated vascular grafts.

Endothelial cell (EC) adhesion, shear retention, morphology, and hemostatic gene expression on fibronectin (FN) and RGD fluorosurfactant polymer (FSP)-coated expanded polytetrafluoroethylene grafts were investigated using an in vitro perfusion system. ECs were sodded on both types of grafts and exposed to 8 dyn/cm(2) of shear stress. After 24 h, the EC retention on RGD-FSP-coated grafts was 59 ± 14%, which is statistically higher than the 36 ± 11% retention measured on FN grafts (p < 0.02). Additionally, ECs on RGD-FSP exhibited a more spread morphology and oriented in the direction of shear stress, as demonstrated by actin fiber staining. This spread morphology has been observed earlier in cells that are adapting to shear stress. Real-time PCR for vascular cell adhesion molecule 1, tissue factor, tissue plasminogen activator, and inducible nitric oxide synthase indicated that the RGD-FSP material did not activate the cells and that shear stress appears to induce a more vasoprotective phenotype, as shown by a significant decrease in VCAM-1 expression, compared with sodded grafts. RGD-FSP-coating allows for a cell layer that is more resistant to physiological shear stress, as shown by the increased cell retention over FN. This shear stable EC layer is necessary for in vivo endothelialization of the graft material, which shows promise to increase the patency of synthetic small diameter vascular grafts.

A novel strategy using cardiac sodium channel polymorphic fragments to rescue trafficking-deficient SCN5A mutations.

BACKGROUND: Brugada syndrome (BrS) is associated with mutations in the cardiac sodium channel (Na(v)1.5). We previously reported that the function of a trafficking-deficient BrS Na(v)1.5 mutation, R282H, could be restored by coexpression with the sodium channel polymorphism H558R. Here, we tested the hypothesis that peptide fragments from Na(v)1.5, spanning the H558R polymorphism, can be used to restore trafficking of trafficking-deficient BrS sodium channel mutations. METHODS AND RESULTS: Whole-cell patch clamping revealed that cotransfection in human embryonic kidney (HEK293) cells of the R282H channel with either the 40- or 20-amino acid cDNA fragments of Na(v)1.5 containing the H558R polymorphism restored trafficking of this mutant channel. Fluorescence resonance energy transfer suggested that the trafficking-deficient R282H channel was misfolded, and this was corrected on coexpression with R558-containing peptides that restored trafficking of the R282H channel. Importantly, we also expressed the peptide spanning the H558R polymorphism with 8 additional BrS Na(v)1.5 mutations with reduced currents and demonstrated that the peptide was able to restore significant sodium currents in 4 of them. CONCLUSIONS: In the present study, we demonstrate that small peptides, spanning the H558R polymorphism, are sufficient to restore the trafficking defect of BrS-associated Na(v)1.5 mutations. Our findings suggest that it might be possible to use short cDNA constructs as a novel strategy tailored to specific disease-causing mutants of BrS.

SCN5A polymorphism restores trafficking of a Brugada syndrome mutation on a separate gene.

BACKGROUND: Brugada syndrome is associated with a high risk of sudden cardiac death and is caused by mutations in the cardiac voltage-gated sodium channel gene. Previously, the R282H-SCN5A mutation in the sodium channel gene was identified in patients with Brugada syndrome. In a family carrying the R282H-SCN5A mutation, an asymptomatic individual had a common H558R-SCN5A polymorphism and the mutation on separate chromosomes. Therefore, we hypothesized that the polymorphism could rescue the mutation. METHODS AND RESULTS: In heterologous cells, expression of the mutation alone did not produce sodium current. However, coexpressing the mutation with the polymorphism produced significantly greater current than coexpressing the mutant with the wild-type gene, demonstrating that the polymorphism rescues the mutation. Using immunocytochemistry, we demonstrated that the R282H-SCN5A construct can traffic to the cell membrane only in the presence of the H558R-SCN5A polymorphism. Using fluorescence resonance energy transfer and protein fragments centered on H558R-SCN5A, we demonstrated that cardiac sodium channels preferentially interact when the polymorphism is expressed on one protein but not the other. CONCLUSIONS: This study suggests a mechanism whereby the Brugada syndrome has incomplete penetrance. More importantly, this study suggests that genetic polymorphisms may be a potential target for future therapies aimed at rescuing specific dysfunctional protein channels.